With the increased understanding of the role of specific cytochrome P450 isoforms in drug metabolism, there is a growing need for improved in vitro methodology using purified P450s. The major drawbacks to current methods include interference from contaminating enzymes present in heterogenous assay systems and difficulties in reconstituting the purified P450s with there associated electron transfer proteins. PanVera proposes to extend technology developed by Dr. Ronald Estabrook for the production of self-sufficient catalytic units composed of P450s linked to NADPH-P450 reductase and to incorporate the fusion proteins into a multi-well format. The goal of the proposed research would be to develop: 1) a panel of P450-reductase fusion proteins, 2) assay methods for absorbance-based detection of substrate metabolism by the fusion proteins. To demonstrate the feasibility of achieving these results, Phase I specific aims will be: 1. Produce and characterize an active recombinant fusion protein of human P450 3A4 linked to human NADPH-P450 reductase. 2. Compare the metabolic profile of the fusion protein with that of the native enzyme system. 3. Examine the extent of coupling between NADPH oxidation and substrate metabolism with the fusion protein. PROPOSED COMMERCIAL APPLICATION: New commercial applications from this research will include: 1) the ability to screen large numbers of test compounds as substrates for specific P450 isoforms, 2) regio and stereospecific synthesis of large amounts of compounds for toxicological, pharmacological and prodrug studies.